Hepatitis C virus (HCV) infection can result in varied clinical outcomes including acute hepatitis, chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Chronic infections are common (>60% of those infected) and an estimated 170 million people are infected worldwide. Despite a great deal of progress, a vast number of unanswered questions remain concerning HCV replication, pathogenesis and immunity. Our long-term goal is to understand, at the molecular level, the roles of viral proteins, RNA elements, and host factors in HCV replication and pathogenesis. Studies on this enveloped plus-sense RNA virus have been (and continue to be) challenging because of its poor replication in cell cultures and the lack of a convenient and inexpensive animal model. Despite this, in our previous studies we have defined the HCV polyprotein processing scheme, examined folding and oligomerization of the viral glycoproteins, characterized two HCV-encoded proteinases in detail, discovered a highly conserved RNA element at the 3' end of HCV genome RNA, and developed an infectious molecular clone of HCV. More recently, we have established efficient cell culture replication and genetic analysis systems for genotype 1a/1b subgenomic and full-length RNAs. Biochemical assays for HCV RNA translation and replication have also been devised. With these tools in hand, this grant focuses on two key players in HCV RNA replication, conserved RNA elements and the NS5A phosphoprotein. RNA elements we will study include a group of novel phylogenetically conserved stem-loop structures in the NS5B coding region. For NS5A, we have previously characterized the NS5A-associated kinase activity, mapped a major phosphorylation site, and studied the effects of various mutations on cell culture adaptation and interferon sensitivity. We will continue our efforts to understand the function of NS5A through additional genetic, biochemical and structural studies.